Synthesis of hyperbranched amphiphilic polyester and theranostic nanoparticles thereof

ABSTRACT

A method of making a hyperbranched amphiphilic polyester compound includes drying under vacuum a mixture of 2-(4-hydroxybutyl)-malonic acid and p-toluene sulphonic acid as catalyst. The vacuum is then released with a dry inert gas after drying. The dried mixture is heated under the inert gas at a temperature sufficient for polymerization. The inert gas is evacuated while continuing to heat the mixture. The formed polymer is then dissolved in dimethylformamide and precipitated out by adding methanol. Modifications of the method yield nanoparticles of polyesters having properties suited for coencapsulating fluorescent dyes together with therapeutic drugs, resulting in theranostic nanoparticles, that is, nanoparticles useful in both therapeutic treatments and diagnostic methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. application Ser. No.13/626,955, filed, Sep. 26, 2012, which is a divisional application ofU.S. application Ser. No. 12/417,017, filed Apr. 2, 2009, entitled“Synthesis of Hyperbranched Amphiphilic Polyester and TheranosticParticles Thereof,” which claims the benefit of U.S. provisionalapplication Ser. No. 61/041,624 filed on Apr. 2, 2008. All of theseapplications are incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grant no CA101781awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of nanotechnology and, moreparticularly, to nanoparticles useful as carriers of fluorescent dyesfor diagnostic purposes and therapeutic drugs for treatment of disease;these dual-purpose particles are also known as “theranosticnanoparticles.”

BACKGROUND OF THE INVENTION

Polymer science has traditionally focused on linear polymers orcross-linked linear polymers, resulting in a wide variety of materialsimplemented in most facets of daily life. Recent progress in polymersciences has resulted in the development of dendrimers1 and mostrecently hyperbranched polymers2-4 consisting of branched structureswith high numbers of reactive groups in their periphery. The synthesesof these multifunctional dendritic (branched) polymers hold greatpromise for targeted delivery of drugs, therapeutics, diagnostics andimaging. The perfectly branched structures called dendrimers areconstructed by an iterative and complex reaction sequence involvingprotection-deprotection steps whereas hyperbranched polymers, the lessperfect structures, are made by one step polymerization reaction. Recentadvances in nonviral drug delivery and cancer chemotherapy have revealedbiocompatible branched polymers like polyethyleneimine (PEI) andstarburst PAMAM as effective drug delivery systems, which can mimicnaturally occurring biological transport systems such as lipoproteinsand viruses.5 Unlike linear polymers which are produced from divalent ABtype monomers, dendritic macromolecules are produced from polyvalent ABnmonomers (n≧2), giving rise to its branching and multiple-endstructures.6-8 Dendritic polymers have gained large interest in recentyears because of their highly branched structures facilitating effectiveencapsulation of guest molecules and having many attractive featuressuch as improved solubility, reactivity, structure architecture,biocompatibility, low viscosity and low crystallinity compared to thoseof linear polymers of same molecular weight.9 Therefore, the creation ofnew and highly branched polymeric nanostructures with multifunctionalcapabilities is central to the development of novel materials withapplications in various fields ranging from drug delivery, immunoassays,microelectrons, coating and nanocomposites.10,11 Polymeric nanoparticlesand nanocomposites with dual fluorescent, magnetic and therapeuticproperties will have a huge impact in medicine, particularly in cancerdiagnosis and treatment, where novel targeted multifunctional polymericnanoparticles can be developed to obtained spatiotemporal informationabout disease stage and progress of a therapeutic regime.12-14 Hence,there has been substantial interest in developing smart therapeutic andselective polymeric vehicles for targeted treatment of various diseases,preventing toxicity to healthy tissues.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides methods for making hyperbranched amphiphilic polyestercompounds. These polyesters may be used to generate nanoparticles havingone or more hydrophobic pockets and a hydrophilic outer surface. Thepolymeric nanoparticles (PNPs) may be used as carriers for a hydrophobicnear-infrared fluorescent dye and/or a therapeutic drug. The PNPs arebiodegradable and, having been modified with appropriate chemical groupsalong their outer surface, are readily taken into cells, thus providingan ideal vehicle for delivery of therapeutic drugs. Since the PNPs maycarry both a fluorescent dye and a therapeutic drug, they can be trackedoptically via the dye and simultaneously deliver the drug topredetermined cells. The capability of having both a therapeuticmodality and a diagnostic modality may be identified as “theranostic.”

A method of the present invention includes making a hyperbranchedamphiphilic polyester compound. The method includes drying under vacuuma mixture of 2-(4-hydroxybutyl)-malonic acid and p-toluene sulphonicacid as catalyst. Then, releasing the vacuum with a dry inert gas afterdrying. The method continues by heating the dried mixture under theinert gas at a temperature sufficient for polymerization. The methodproceeds by evacuating the inert gas while continuing to heat themixture, then dissolving the formed polymer in dimethylformamide.Finally, the method ends after precipitating the dissolved polymer byadding methanol.

In the method, drying may comprise a mixture of2-(4-hydroxybutyl)-malonic acid and p-toluene sulphonic acid inapproximately a 100:1 molar ratio. Also, drying under vacuum preferablycomprises a high vacuum and the inert gas is argon gas. The heating ispreferably at a temperature of approximately 150° C., which promotespolymerization. The heating may continue for approximately two hours.Evacuating is most preferably conducted slowly at approximately 0.2mm/Hg for about one hour while maintaining the polymerizationtemperature. After polymerization, the method may further comprisepurifying the polymer by separating the precipitate, washing it withmethanol and drying it in a vacuum.

The described method may be modified to make aminated PNPs. This isaccomplished by dissolving the precipitated polymer in anhydrousdimethylformamide (DMF), adding 1,1′-carbonyldiimidazole drop-wise toform a reaction mixture and incubating the reaction mixture at roomtemperature for approximately one to two hours. This method continues byadding ethylenediamine in anhydrous DMF drop-wise and continueincubation of the reaction mixture at room temperature for approximately24 hours, then precipitating the reaction mixture in methanol,separating the precipitate and drying in a vacuum to obtain a purifiedhyperbranched polyester amine.

Yet another modification of the described method is useful for makingpropargylated PNPs. This modification includes dissolving theprecipitated polymer in anhydrous dimethylformamide (DMF), adding1,1′-carbonyldiimidazole drop-wise to form a reaction mixture,incubating the reaction mixture at room temperature for approximatelyone to two hours, then adding propargyl chloride in anhydrous DMFdrop-wise and continue incubation of the reaction mixture at roomtemperature for approximately 24 hours. Lastly, the method calls forprecipitating the reaction mixture in methanol, separating theprecipitate and drying in a vacuum to obtain a purified hyperbranchedpropargylated polyester amine.

Having described the method and its two modifications, the polymersgenerated thereby represent novel molecules useful at least for makingthe PNPs of the invention. Accordingly, the invention includes a polymercomprising the repeating unit HBPE (5).

The invention additionally includes a polymeric nanoparticle comprisingthe polymer HBPE(5), the nanoparticle also having a hydrophobicnear-infrared fluorescent dye encapsulated therein. The dye may beselected from the group consisting of DiI, DiR, and DiD. Additionally,this PNP may include a therapeutic drug coencapsulated with saidfluorescent dye and, particularly, an anti-cancer drug such asazidothymidine.

Another polymer included in the invention is one comprising therepeating unit HBPE-EDA (6).

Moreover, the invention further includes a polymeric nanoparticlecomprising the polymer HBPE-EOA (6) and a hydrophobic near-infraredfluorescent dye encapsulated therein. As noted above, the dye may beselected from the group consisting of DiI, DiR, and DiD and the PNP mayalso include a therapeutic drug coencapsulated with said fluorescentdye.

The other modification of the presently disclosed method is useful formaking a polymer comprising the repeating unit HBPE-PA (7), as set forthbelow.

Included in the invention is a polymeric nanoparticle comprising thepolymer HBPE-PA (7) and a hydrophobic near-infrared fluorescent dyeencapsulated therein. In this polymeric nanoparticle the dye may beselected from the group consisting of DiI, DiR, and DiD, and there mayalso be a therapeutic drug coencapsulated with said fluorescent dye. Thetherapeutic drug preferably comprises an anti-cancer drug, for example,azidothymidine or wherein the anti-cancer drug comprises paclitaxel.

Those skilled in the art will recognize that while certain hydrophobicnear-infrared fluorescent dyes have been given as examples, other dyeshaving similar properties would also be useful in the invention. Thesame can be expected to hold for therapeutic drugs other than the onesgiven here as examples; as long as the drug exhibits sufficienthydrophobicity to nest in the hydrophobic pocket formed by the polymerin the nanoparticle, the drug should be of use in the invention. Thesedyes and drugs as known to the skilled by their properties are,therefore, intended to be included within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1 illustrates, according to a method of the invention, the facilesynthesis of amphiphilic hyperbranched polyester (HBPE, 5) andcorresponding polyester-amine (HBPEA, 6) polymer, which are highlybranched, globular, and biodegradable in nature; the polymeric backboneof HBPE was synthesized by melt polymerization of the monomer 4, whichcan be easily made from commercially available diethylmalonate 1 andbromobutyl acetate (2) in two simple steps, hence the synthesis of 5 iscost-effective; the corresponding aminated polymer can be synthesizedvia conjugation of ethylenediamine using 1,1′-carbonyldiimidazile (CDI)coupling; the resulting amphiphilic and dendritic polymers (5 and 6)contain inner hydrophobic domain (aliphatic chains), surrounded by ahydrophilic outer shell (carboxylic or amine groups), which after selfassembly in water result in a stable nanoparticle suspension which canencapsulate hydrophobic dyes and drugs;

FIG. 2 shows a schematic representation of the structure of dendriticpolyester (HBPE 5) and corresponding formation of polymericnanoparticles via solvent diffusion method; using this method, a seriesof hydrophobic drugs and dyes can be encapsulated in one pot; thesepolymeric nanoparticles are highly dispersed in water and stable in awide range of buffered solutions under physiological conditions; bothclick chemistry, carbodiimide chemistry and other conjugationchemistries can be used for the functionalization of these polymericnanoparticles with small molecule like antibodies, proteins,oligonucleotides and other targeting agents to generate ananoparticle-ligand library;

FIG. 3 presents an overall schematic representation of the preparationof functional polymers and polymeric nanoparticles (PNPs); polymer 5 wassynthesized following the melt polymerization technique; PNPs weresynthesized using the solvent diffusion method and were stable in waterand other aqueous buffer solutions; carbodiimide chemistry has beenfollowed for the synthesis of functional polymers (6 and 7) using1,1′-carbonyldiimidazole (CDI), as a water insoluble carbodiimide. NearIR dyes (DiI, DiD and DiR), pacitaxel and AzT encapsulated PNPs wereprepared in water from the water insoluble functional polymers using thesolvent diffusion method. Click chemistry and carbodiimide chemistry hasbeen used for the synthesis of a library of functional PNPs; insets showthe structures of NIR dyes, Paclitaxel and AzT;

FIG. 4 is a representation of the synthesis of azide-functionalizedfolic acid (13) from chloropropylamine and CuI-catalyzed “click”chemistry for the preparation of folate-functionalized PNPs (11a-e);

FIG. 5 shows ¹H NMR spectrum of the AB2 monomer (3); the characteristictriplet for the single acidic proton (f) was observed at 3.34 ppm;

FIG. 6 depicts a ¹³C NMR spectrum of the AB2 monomer (3); all peaks areassigned to the corresponding carbons, which confirms the presence ofthe expected compound;

FIG. 7 shows ¹H and ¹³C NMR spectra of the final AB2 monomer (4) usingCDCl₃ as a solvent; absence of the ethyl ester protons and acetyl groupin the NMR spectra confirms the hydrolysis of compound 3 and theformation of compound 4; all peaks are assigned to the correspondingprotons and carbons, which confirm the presence of the expected product;

FIG. 8 shows ¹H and ¹³C NMR spectra of amphiphilic hyperbranched polymer(5), confirming the preparation of high molecular weight polymer; thebroadening of the sharp peaks in ¹H NMR spectrum of the monomers alsoindicates the formation of polymer having a large number of chemicallyequivalent protons with same δ value;

FIG. 9 provides GPC traces of polyester 5, polymerized at 150° C. atatmospheric pressure and under high vacuum; a) before applying vacuum,showing the presence of low molecular weight polymers and oligomers; b)high molecular weight polymer was formed after applying vacuum; for acomparative study between the average molecular weight (Mw) and thepolymerization time at 150° C., the samples were taken from the reactionmixture periodically and analyzed by GPC; with increasing time there wasan increase in the molecular weight, whereas a dramatic increase in thepolymer's molecular weight was observed when high vacuum was applied;moreover, when the evacuation was continued for more than 2 h, theresulting polymers were found to be insoluble in all the solvents; theaverage molecular weight of polymer (5) was Mw=42,000, PD=1.6;

FIG. 10 is a TGA thermogram trace of polyester 5 at a heating rate of10° C./min in air; the thermogram obtained is typical of an aliphatichyperbranched biopolymer; the hyperbranched polymer exhibits a moderatethermal stability; thermal decomposition of the polymer was initiated at˜210° C.; at 225° C., the polymer has only lost about 2.5% of itsweight, mainly due to evaporation of the volatile compounds (such asH2O, CHCl3 and DMF), before the induction of its thermal degradation;approximately a 10% weight loss occurred at 250° C.;

FIG. 11 shows in A) the chemical structure of DiD, DiI and DiR; thesedyes are water insoluble in nature and are primarily used to visualizecell membranes; and in B) a photographic image showing an aqueous (PBS)suspension of HBPE nanoparticles encapsulating the corresponding dyes(8a-c); similarly, one can encapsulate a hydrophobic drug or acombination of drug and dye; highly dispersed dyes/drugs encapsulatedpolymeric nanoparticles are stable in wide range of solvents underphysiological conditions. The fluorescence of the resultingdye-encapsulated polymeric nanoparticles is bright and is notaccompanied by any significant quenching upon encapsulation ofphotobleaching upon prolonged imaging;

FIG. 12 shows the hydrodynamic diameters of the nanoparticles asmeasured by dynamic light scattering (DLS) instrument; measurement datashows the average hydrodynamic diameter of the particles are rangingbetween approximately 90±20 nm;

FIG. 13 presents Scanning Electron Microscope (SEM) images of thepolymeric nanoparticles showing an average diameter ranging from 115±25nm, in accordance with the DLS data shown in FIG. 12; remarkably, theblack rectangular box in the image indicates that the nanoparticles arespherical in shape, as expected;

FIG. 14 depicts FT-IR spectra of the final monomer 4 (Acid), HBPE 5(Polymer) and the dye encapsulating PNPs 8a, demonstrating presence ofdye within the PNPs; the presence of a FT-IR band at 1728 cm⁻¹ for theester group indicates the formation of the hyperbranched polyester fromthe monomer (band at 1710 cm⁻¹ for aliphatic carboxylic acid group); theband at 1675 cm⁻¹ is attributed to a conjugated alkene group, confirmingthe encapsulation of the dye inside a hydrophobic cavity of the PNPs;

FIG. 15 are UV/Vis spectra of the dye (DiI, DiD, DiR)-encapsulating PNPs(8a-c), showing the presence of the NIR dyes with absorption maxima at552, 650 and 755 nm, respectively;

FIG. 16 shows the characteristic fluorescence emission spectra of dyeencapsulating polymeric nanoparticles (8a-c) in DI water; thefluorescence intensity maxima of these nanoparticle solutions at 570 nm,675 nm and 780 nm indicate the presence of NIR dyes DiI, DiD and DiR,respectively, in the hydrophobic domain of the polymeric nanoparticles;no changes in fluorescence intensity or quenching of the dyes wasobserved upon encapsulation and subsequent storage of the nanoparticlesat 4° C. for months, demonstrating the high fluorescence stability ofthese dye encapsulated polymeric nanoparticles; note the multipleimaging capability using 3 different wavelengths;

FIG. 17 are UV/Vis spectra of the DiI dye encapsulating PNPs (8a) andthe dye alone; a blue shift (by 10 nm) was observed in the UV/Visabsorption maxima in the case of DiI encapsulating PNPs, which confirmedthe presence of the dye within the hydrophobic domain of the PNPs;

FIG. 18 depicts the blue shift (23 nm) in the fluorescence emission ofDiI encapsulating HBPE nanoparticles (8a) as compared to free dye; thisis due to both van-der Walls (hydrophobic) and electronic interactionsof the dye with the polymeric cavity, confirming the presence of dyeinside the polymer cavity;

FIG. 19 shows a photo-stability study of the DiI encapsulating PNPs (8a)and DiI alone in solution in the presence of UV light, demonstrating thestability of the dye when encapsulated inside the polymeric cavitycompared to the free dye;

FIG. 20 shows the electrokinetic potential (zeta potential) of thesynthesized polymeric nanoparticles; the zeta potential of thenon-aminated polymeric nanoparticles is negative (A, ζ, =−54.5 mV), asexpected, due to the presence of surface carboxylic acid groups; notsurprisingly, the zeta potential of the aminated polymeric nanoparticlesis positive (B, ζ, =10.33 mV), where the low positive value indicatesthe partial amination of the nanoparticles with the presence of lessnumber of free carboxylic groups than amine groups at the surface;

FIG. 21 presents cell internalization studies using Xenogen's IVIS 50;in these experiments either carboxylated (negative, 8b) or aminated(positive, 9b) nanoparticles were incubated with cells from the A549lung cancer cell line; in these representative studies, near infraredDiR encapsulated nanoparticles were used, although similar results wereobserved with DiD and DiI; as expected, internalization was observedonly when the positively charged aminated DiR nanoparticles were used,as judged by the near infrared fluorescence coming from the cell pelletson FIG. 21B; note that when carboxylated nanoparticles are used (FIG.21A), the fluorescence remains in the supernatant; these resultsdemonstrate that cationic nanoparticles are a better candidate for thecell internalization studies and for cell tracking studies. In addition,it shows the capability of imaging in the near infrared;

FIG. 22 is a UV/Vis spectrum of the folate-clicked, DiI-encapsulatingPNPs a, showing the presence of both the encapsulated dye (553 nm) andsurface clicked with folate (354 nm); similar results were obtained forthe paclitaxel- and DiI-encapsulating PNPs 11d, prepared for targetedcancer therapy;

FIG. 23 shows an UV/Vis spectrum of the folate-clicked,DiR-encapsulating PNPs 11b, showing the presence of both theencapsulated DiR (755 nm) and surface clicked with folate (355 nm);

FIG. 24 shows bar graphs acknowledging the potential biomedicalapplications of the synthesized PNPs; we evaluated their cytotoxicity,through the MTT assay; first, we examined the potential in vitrodifferential cytotoxicity of carboxylated, aminated, andfolate-decorated DiI-containing PNPs, using a lung carcinoma (A549) cellline; results indicated that the carboxylated and folate-conjugated PNPsexhibited nominal cellular cytotoxicity (less than 4% compared to thecontrol), whereas the aminated PNPs induced cell death to approximately10% of the cell population (FIG. 24 a); indeed, the folate-decorated DiIand paclitaxel co-encapsulating PNPs (11d) induced a significantreduction in cell viability, as more than 50% of the cell populationunderwent cell death (FIG. 24 b); furthermore, as most lung carcinomasexhibit aberrant telomerase activity, leading to cell immortality, weencapsulated the reverse transcriptase inhibitor AzT in PNPs (11e); wefound that folate-decorated DiI and AzT co-encapsulating PNPs inducedsignificant cell death, as previously reported in literature viainhibition of telomerase activity (FIG. 2 b); overall, these datasuggest that the induction of cell death is mainly mediated by eitherpaclitaxel or AzT, and not by the fluorophores; control cells weretreated with 1×PBS; average values of four measurements are depicted±standard error;

FIG. 25 shows a confocal laser-scanning microscopic image of A549 lungcancer cells incubated with DiI dye-encapsulated carboxylated polymericnanoparticles (8a); dye encapsulated nanoparticles are incubated withthe cells for 6 h; result shows no internalization of the nanoparticlesinto the cytoplasm, which demonstrates that the anionic (carboxylicgroups at surface) polymeric nanoparticles are not the appropriatecandidate for cell internalizations; instead, only cell membrane stainedwith the dye (outer red lines); the nucleus stained with DAPI (bluecolor);

FIG. 26 shows a confocal laser-scanning microscopic image of A549 lungcancer cells incubated with DiI dye encapsulated aminated polymericnanoparticles (9a); internalization of the nanoparticles into the cellcytoplasm was observed, which demonstrates that the cationic (aminegroups at surface) polymeric nanoparticles are the appropriate candidatefor cell internalizations; the nucleus stained with DAPI (blue color);

FIG. 27 depicts a confocal laser-scanning microscopic image of A549 lungcancer cells incubated with DiI dye encapsulated folate-immobilizedpolymeric nanoparticles (11a); the particles were incubated with thecells for 6 h.; internalization of the nanoparticles into the cell wasobserved, demonstrating the presence of folate receptor in the A549cancer cells and therefore inducing a folate-receptor mediatedinternalization;

FIG. 28 provides a confocal laser-scanning microscopic image of A549lung cancer cells incubated with folate modified nanoparticles (11d)encapsulating both a hydrophobic dye (DiI) and a hydrophobic anti-cancerdrug (paclitaxel); the nanoparticles were incubated with the cells for 6h. Neither the fluorescence intensity of the dye, nor the cytotoxiceffects of the anti-cancer drug are affected when encapsulated in thepolymeric nanoparticle; experiments show that paclitaxel-induced mitoticarrest results in apoptotic cell death of lung carcinoma cells (A549);

FIG. 29 is a confocal laser-scanning microscopic image of A549 lungcancer cells incubated with folate modified nanoparticles (11e)encapsulating both a hydrophobic dye (DiI) and a hydrophobic anti-HIVdrug (AzT); the nanoparticles were incubated with the cells for 6 h;these experiments show that AzT-induced mitotic arrest results inapoptotic cell death of lung carcinoma cells (A549);

FIG. 30 provides an assessment of the PNP-cell association via flowcytometry, where in a) absence of fluorescence emission is observed incontrol mock-treated cells (1×PBS), in b) partial association of thedye-loaded non-aminated PNPs (8a) is observed, in c) aminated (9a) andd) folate-decorated (11a) nanoparticles interact more profoundly withthe cells, as indicated by higher levels of fluorescence emission; and

FIG. 31 shows drug (paclitaxel) and dye (DiI) release profiles offunctional PNPs (1d) in PBS (pH=7.4) at 37° C.; release of paclitaxel (A& B) and DiI (C & D) were observed in the presence of an esterase enzyme(A & C) and at pH 4.0 (B & D); these results indicate that the PNPs aredegradable in the presence of an esterase enzyme and at low pH; acontrolled release of drug and dye was observed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Anypublications, patent applications, patents, or other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

Herein, we report the synthesis of novel biodegradable hyperbranchedpolyesters and their use for the synthesis of cell-permeable polymericnanoparticles that encapsulate hydrophobic dyes and drugs for dualoptical imaging and therapeutic applications. To this date, no one hasmade biocompatible polymeric nanoparticles from diethylmalonate basedhyperbranched polyester. The design and synthesis ofdiethylmalonate-based AB₂ monomer is novel and tuned in such a way thatthe resulting polymer will have three-dimensional molecular architecturewith hydrophobic interior and hydrophilic segments at the surface(amphiphilic). Selective mono-C-alkylation of diethylmalonate using amild basic condition and followed by hydrolysis of the monomer wasperformed to develop a new, water-soluble AB₂ monomer for the synthesisof the hyperbranched polyester. We have employed a melt polymerizationtechnique using para-toluenesulfonic acid [p-TSA] as a catalyst tosynthesize the novel aliphatic and biodegradable hyperbranchedpolyester. We hypothesized that the presence of AB₂ branching point anda hydrophobic butyl chain in the monomer structure could be able togenerate a highly branched and hydrophobic polymer. As aproof-of-principle, the resulting polyester was highly branched,amphiphilic, having carboxylic acid groups at the surface and obtaininga three dimensional architecture with hydrophobic cavity. Therefore,compare to the conventional linear polymers, our branched polyester isamorphous, amphiphilic, soluble, biodegradable, highly surfacefunctional and has cavities for effective encapsulation of guestmolecules, which suggest its versatility in biomedical applications.Post-functionalization of this water insoluble polyester has been doneusing carbodiimide chemistry resulting in cationic and clickablehyperbranched polyester.

A solvent diffusion method has been adopted for the synthesis ofpolymeric nanoparticles (PNPs) where the hydrophobic areas assembletogether to minimize contact with the aqueous environment, whileexposing the hydrophilic segments containing carboxylic groups at thesurface in aqueous solution.^(15,16) This results in the formation ofcarboxyl functionalized spherical polymeric nanoparticles in watercontaining inner hydrophobic domains that can encapsulate hydrophobicmolecules such as dyes and drugs.¹⁷ Note that, this is the first exampleof development of hyperbranched polyester based polymeric nanoparticlesusing solvent diffusion method. Experimental data showed the effectiveencapsulation of various hydrophobic near infrared (NIR) dyes and atherapeutic drug without significant precipitation or reduction of thefluorescent properties. The fluorescence of the resulting PNPs is brightand stable, allowing the imaging of cells without significantphoto-bleaching. Click chemistry has been used for the synthesis offolate decorated PNPs for the targeted cancer therapy.⁸⁰⁻²⁰ Finally, wehave been able to encapsulate either a hydrophobic antitumor drug(Paclitaxel) or, a nucleoside analog reverse transcriptase inhibitor(AzT) for the treatment of HIV and AIDS, along with near infra redfluorescent dyes (DiI or DiR) into the folate decorated PNPs fortargeted drug delivery and imaging. We have used human lung carcinoma(A549) and normal cardiomyocites (h9c2) cell lines throughout all invitro studies. We have assessed MTT assay to determine the cytotoxity ofour functional PNPs. Results showed Taxol® and AzT encapsulated PNPswere toxic to the cancerous cell lines, whereas, dye encapsulatedpolymeric fluorophores were non-toxic. These results were corroboratedwith confocal microscopic studies and FACS analysis. The PNPsdegradation and controlled drug and dye release experiments wereperformed under enzymatic and low pH environments. Most importantly, weare successful in animal imaging using mice model, in vivo, with the NIRdye (DiR and DiD) encapsulated PNPs for animal imaging applications.

Therefore, our present protocol is capable of creating a library ofmultifunctional therenostic (therapeutics and optical diagnostics)polymeric nanoparticles for biomedical applications including (a)encapsulated chemotherapeutic agents (Taxol® and AzT) for HIV and cancertherapy, (b) surface functionality (folic acid ligand) for cancertargeting, (c) “click”-chemistry-based conjugation of targeting ligands,(d) encapsulated NIR dyes for fluorescent imaging capabilities and (e)thermomechanical applications including luminescent, conductive,magnetic or radioprotection of the corresponding polymeric-metallicnanocomposites.

Results and Discussion Synthesis and Characterization of BiodegradableHyperbranched Polymers

The amphiphilic hyperbranched polyester (HBPE 5) was rationally designedfor the development of theranostic PNPs (nanoparticles providing both atherapeutic agent and a diagnostic modality), by employing strategies ofnanoparticle formation and drug/dye encapsulation in one process. FIG.1-2 shows our synthetic strategy, leading to the formation of a novel,water soluble, AB₂ monomer 4 which upon polymerization gives rise to thewater insoluble, biodegradable polymer 5, capable of encapsulatingdyes/drugs for therapeutic applications. The melt polymerizationtechnique was followed for the polymer synthesis, where we observed thatat the initial stages of polymerization, oligomers and low molecularweight polymers were obtained. However, upon applying vacuum, a highmolecular weight polymer (M_(w)=42,000, PD=1.6) was formed (FIG. 9). Theresulting polyester was highly branched, having carboxylic acid groupsat the surface and obtaining a three dimensional architecture withhydrophobic cavity. Hence contrary to conventional linear polyesters,our branched polymer is amorphous, amphiphilic, soluble, highly surfacefunctional, biodegradable and has a cavity for effective encapsulationof guest molecules, which suggest its versatility in biomedicalapplications. Through thermal gravimetric analysis (TGA), we determinedthat the polymer exhibits moderate thermal stability (10% weight loss at250° C. in air), which is typical for a biodegradable polymer (FIG. 10).The polymer was further characterized using spectroscopic andchromatographic techniques (FIG. 5-8). Subsequently, the presence offree carboxylic acid groups at the surface prompted the generation of alibrary of functional polymers using carbodiimide chemistry. We used1,1′-carbonyldiimidazole (CDI), as a water insoluble carbodiimide, andeither ethylenediamine or propargylamine for the synthesis ofsurface-aminated cationic polymer (HBPE-EDA 6) and clickable polymer,respectively (HBPE-PA 9, FIG. 3). Hence, the former surface-aminatedpolymer may be a good candidate for non-specific cell internalization,whereas, the later one might be a platform for targeted therapy due tothe facile conjugation of specific cellular receptor ligands, such asfolic acid, via click chemistry (FIG. 4).

Polymeric Nanoparticle (PNP) Synthesis and Drug/Dye Encapsulation

In order to prepare functional PNPs, a modified solvent diffusion methodwas used, where the nanoparticle formation and guest moleculeencapsulation in the hydrophobic cavity took place in one-pot. Theamphiphilic polymer and hydrophobic guests were dissolved in anhydrousdimethylformamide (DMF) and added drop-wise to water under continuousstirring, driving both the self-assembly and encapsulation processes andresulting in the synthesis of functional PNPs. The resulting PNPs werehighly stable in aqueous buffered solution for more than a year, withoutsignificant reduction in the fluorescent emission of the encapsulateddyes and can be concentrated without significant precipitation.Therefore, near infra red dye (DiI, DiR and DiD) encapsulated PNPs (8a-cand 9a-c) were synthesized from the corresponding carboxylated andaminated polymers (HBPE 5 and HBPE-EDA 6, respectively). Alternatively,the aminated PNPs (9a-c) can be prepared from the carboxylated PNPs(8a-c) using water soluble carbodiimide, EDC,[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] andethylenediamine (FIG. 3). Amination of these nanoparticles was confirmedby an overall surface charge (4-potential) measurement, where a positivesurface potential was obtained in the case of aminated PNPs (FIG. 20).Propargylated PNPs were also synthesized with DiI dye (10a), DiR dye(10b), DiD (10c), DiI with the anti-cancer drug paclitaxel (10d) andwith AzT (10e) using the above strategy. These PNPs (10a-e) are veryimportant synthon for the synthesis of a library of functional PNPs viaclick chemistry. To demonstrate the applicability of click chemistry inthis system, the alkyne-azide click was engineered to occur at theinterface between the propargylated carboxylic acid corona of the PNPsand the aqueous phase in which the azide-functionalized folic acid isdissolved. Therefore, folate-decorated PNPs (11a-e) were prepared usingthis 1,3-dipolar cycloaddition reaction, click chemistry, mediating thetargeted drug delivery to cancer cells that overexpress the folatereceptor (FIG. 4).

Polymeric Nanoparticle Characterization

The approximate hydrodynamic diameter of the PNPs was determined thoughDynamic Light Scattering (DLS), ranging from 100±20 nm, which wassimilar to that of the unmodified nanoparticles (FIG. 12). Well-formedspherical PNPs were observed by scanning electron microscopy (SEM, FIG.13) and the average diameter of these PNPs was 115±25 nm, demonstratinga direct correlation with the DLS data. Remarkably, the blackrectangular box in the SEM image indicates that the shape of thenanoparticles was spherical as expected from the structure of theoriginal polymer HBPE 5. The formation of hydrophobic microdomains andthe encapsulation of NIR dyes inside the PNPs were confirmed byfluorescence measurement of free DiI dye and encapsulated DiI in PNPs(8a, FIG. 18). In this experiment, DiI dye was dissolved in DMF andallowed to evaporate at room temperature and then dispersed in water. Ablue shift (by 23 nm) was observed in the fluorescence emission ofDiI-encapsulated HBPE nanoparticles (570 nm) as compared to the freenon-encapsulated DiI dye (593 nm) in water. This indicated the presenceof the dye inside the electronic environment of the polymer's cavity,confirming the presence of hydrophobic microdomains in the PNPs. Similarresults were obtained from UV/Visible spectroscopy studies, where a blueshift was observed for the encapsulated DiI (FIG. 17), furtherconfirming the entrapment of the dye inside the cavity. Interestingresults were obtained from Fourier Transform Infra Red (FT-IR)spectroscopy (FIG. 14), and these corroborated the formation of PNPsencapsulating the DiI dye. The presence of the aliphatic alkenes'characteristic band at 1675 cm⁻¹ indicated the encapsulation of DiI viaconjugated double bonds inside the nanoparticle cavity. Subsequently, weprepared three different dye (DiI, DiR and DiD) containing PNPs (8a, 8band 8c, respectively) from the polymer 5 as shown in FIG. 3 and FIG. 15.Characteristic fluorescence emission spectra of the dye-loaded PNPs inPBS buffer are shown in FIG. 16. The fluorescence intensity maxima ofthese nanoparticles were at 570, 675 and 780 nm, indicating the presenceof the NIR dyes DiI, DiD and DiR, respectively, within the hydrophobicdomain of the PNPs. These functional fluorophore-containing PNPs werehighly stable in aqueous solution (FIG. 11). Next, NIR dye andpaclitaxel co-encapsulated folate-clicked PNPs (11a-e) werecharacterized by UV/Vis spectroscopy (FIG. 22-23).

In Vitro Cytotoxicity

Having in mind the potential biomedical applications of the synthesizedPNPs, we evaluated their cytotoxicity, through the MTT assay. First, weexamined the potential in vitro differential cytotoxicity ofcarboxylated, aminated, and folate-decorated DiI-containing PNPs, usinga lung carcinoma (A549) cell line. Results indicated that thecarboxylated and folate-conjugated PNPs exhibited nominal cellularcytotoxicity (less than 4% compared to the control), whereas theaminated PNPs induced cell death to approximately 10% of the cellpopulation (FIG. 24 a). Considering these findings, the potential use ofthese PNPs in imaging and drug delivery applications, either in vitro orin vivo, is anticipated. Hence, we examined if PNPs can be used fortargeted drug delivery, by examining the cytotoxic efficacy of PNPsco-encapsulating DiI and the hydrophobic chemotherapeutic agentpaclitaxel. Indeed, the folate-decorated DiI and paclitaxelco-encapsulating PNPs induced a significant reduction in cell viability,as more than 50% of the cell population underwent cell death (FIG. 24b). Furthermore, as most lung carcinomas exhibit aberrant telomeraseactivity, leading to cell immortality, we encapsulated the reversetranscriptase inhibitor AzT in PNPs. We found that folate-decorated DiIand AzT co-encapsulating PNPs induced significant cell death, aspreviously reported in literature via inhibition of telomerase activity(FIG. 24 b). Overall, these data suggest that the induction of celldeath is mainly mediated by either paclitaxel or AzT, and not by thefluorophore. Specifically, the enhanced in vitro cytotoxicity of thefolate-decorated DiI and paclitaxel co-encapsulating PNPs in A549 cellshints the successful targeting of carcinomas that overexpress the folatereceptor on their plasma membrane. Furthermore, these results suggestthat the co-encapsulation of a fluorophore and a therapeutic agent inour PNPs can be utilized for cellular targeting, such as in cancer oranti-HIV CD4′-specific therapeutic regimes, visualization of the drug'shoming and monitoring of tumor regression in clinical studies.

In Vitro Cellular Uptake of PNPs

To demonstrate the capability of our functional PNPs to be internalizedby cells and eventually exert specific intracellular activity, variouspreparations of PNPs were incubated with lung carcinoma cells (A549) for6 h. Confocal images showed there was no internalization of thenon-aminated PNPs, but only the cell membranes were found to be stainedwith the red Dil dye (outer red lines, FIG. 25). This demonstrates theproof-of-concept that the anionic (carboxylic groups at surface) PNPsare not appropriate candidates for cell internalization. To furthercorroborate this concept, we used A549 cells treated with aminated PNPsencapsulating Dil (9a). Contrary to the carboxylated PNPs, there wasstrong internalization of the cationic surface PNPs (FIG. 26). Notably,these PNPs did not affect cellular integrity and nuclear stability,failing to trigger apoptosis, even after 12 h of incubation. Hence,these results strongly support the notion that cationic (surface aminegroups) PNPs are better vehicles for cell internalization. Subsequently,we performed in vitro cellular uptake studies, utilizing a Xenogen IVISsystem. Similar results to the confocal studies were obtained.Specifically, aminated PNPs were found intracellularly, as fluorescentemission was recorded from the cell pellet. On the other hand, there wasabsence of fluorescence emission from the pellet of cells treated withnonaminated PNPs, suggesting lack of PNP internalization (FIG. 21, IVISimages). Similar results were obtained from other synthesized PNPs (8b-cand 9b-c) through the IVIS setup, in line with the confocal microscopyobservations.

Then, we investigated the targeting potential of our PNPs and cellularuptake of the folate-clicked PNPs (11a), comparing these PNPs with thecorresponding carboxylated ones (8a). Confocal microscopy revealed theeffective uptake of the folate-functionalized PNPs by A549 cells (FIG.27), in contrast to the carboxylated ones (FIG. 25). The enhancedcellular uptake of the folate-decorated PNPs may be attributed tofolate-receptor mediated internalization. Accordingly, through confocalstudies, we observed that the efficiency of folate-decorated PNPs uptakesignificantly improved upon increasing the incubation time, while nocytotoxic effects were observed via the MTT assay. Hence, the enhancedtime-dependent uptake and retention of these PNPs is likely due tofolate-receptor recycling, which is typical of constitutive nutrientreceptor endocytic trafficking. Subsequently, to demonstrate thefolate-clicked PNPs' (11a) proof-of-concept theranostic capabilitytowards cancer cells, we used DiI and paclitaxel co-encapsulating PNPs(10d). Then, the surface propargyl groups were clicked withazide-functionalized folic acid, in order to achieve targeted drugdelivery with optical imaging capability for spatiotemporal monitoring.Lung carcinoma cells overexpressing the folate receptor were treatedwith these PNPs (11d). After a 3 h-long incubation, confocal microscopicexamination revealed cellular internalization and induction ofpaclitaxel-mediated mitotic arrest (FIG. 28), in accordance to theliterature. This illustrates that paclitaxel's therapeutic efficacy waspreserved, despite its PNP encapsulation. Furthermore, treatment withpaclitaxel-containing PNPs triggered dramatic cellular morphologicalchanges after 12 h of incubation, leading to cell death. Furthermore, asmost lung carcinomas exhibit aberrant telomerase activity, leading tocell immortality, we encapsulated the reverse transcriptase inhibitorAzT in PNPs. We found that folate-decorated DiI and AzT co-encapsulatingPNPs induced significant cell death, as previously reported inliterature via inhibition of telomerase activity (FIG. 29). Theseobservations strongly support the importance of encapsulating thispotent anti-tumor agent within the polymeric cavity and targeting itsdelivery, in order to prevent damaging non-transformed cells and healthytissue. Taken together, these findings support the principle thatfolate-decorated PNPs can target and deliver chemotherapeutic agents tofolate-receptor-overexpressing carcinomas, while visualizing the drug'shoming. Thus by modifying the targeting moiety at the theranostic PNPs'surface, other carcinomas or ailing cells may be targeted tailoring thetherapeutic regime, while obtaining important spatiotemporal informationfor clinical decision making.

Flow Cytometric Assessment of PNPs Uptake

To corroborate the PNPs cellular uptake ability, a detailed flowcytometry analysis was performed with functional PNPs (8a, 9a and 11a)and A549 cells. Specifically, through flow cytometry, we determined theDiI-derived cell-associated fluorescence emission in a quantifiablefashion. As shown in FIG. 30 b, limited fluorescence emission wasobserved from cells treated with the carboxylated PNPs (8a). Thisindicated nominal cell association of these carboxylated PNPs (8a), whencompared to the control non-treated cells (FIG. 30 a) where there waslack of fluorescence emission. This is in accordance with the data fromthe confocal and IVIS studies, confirming the observation that theanionic surface of the nanoparticles interacts with the cell's plasmamembrane. Contrary to this and similar to the confocal microscopicobservations, cells incubated with aminated PNPs (9a) showed three-foldhigher fluorescence emission and binding activity when compared to thecontrol mock-treated cells, as shown in FIG. 30 c. Similar to othercationic small molecules and peptides, the interaction of thesurface-localized positive charge of the aminated PNPs with thenegatively charged cell membrane facilitated the association of the PNPswith the cell membrane at the extracellular milieu and the subsequentcellular uptake and retention, as observed through previously discussedin vitro studies. Interestingly, upon clicking the carboxylated PNPswith folic acid, a higher cell-associated fluorescence emission wasobserved (FIG. 30 d). Notably, the profound cellular uptake of thefolate-decorated PNPs (11a), being comparable to the aminated ones, isattributed to the specific folate-receptor-mediated internalization andintracellular retaining. Overall this indicates that the specifictargeting of PNPs through targeting moieties, such as folate, isfeasible and equally efficient as the non-specificelectrostatic-mediated uptake of cationic entities, rendering targetedPNPs useful for potential in vivo applications.

Drug/Dye Release Study of Functional PNPs

The therapeutic application of our polymeric nanoparticles is influencedby the rate of release of the encapsulated drug from the polymericcavity. To evaluate 11d's drug release profile, enzymatic (esterase) andlow-pH degradation experiments were performed. Results indicate a fastrelease of the drug (paclitaxel) from the nanoparticle 11d upon esteraseincubation, reaching a plateau within 4 hours (FIG. 31A). A similarrelease profile of the drug was observed at pH 4.0, reaching a plateauwithin 4.5 hours (FIG. 31B). No significant release of the drug wasobserved from nanoparticles incubated in PBS, pH 7.4. These resultsdemonstrate the stability of the polymeric nanoparticles during storage(PBS), and their cargo release only after cellular uptake via eitheresterase-mediated degradation or in acidified lysosomes. Only afterfolate-receptor-mediated uptake did the PNPs 11d become cytotoxic uponintracellular release of the therapeutic agent. Interestingly, evenslower release of the dye was observed, both upon esterase incubationand at pH 4.0 (FIGS. 31C and 31D). However, no release of the dye wasobserved at normal physiological pH (7.4). The observed differentialrelease of the drug vs. the dye from PNPs 11d may be attributed to thedrug's (paclitaxel) size and hydrophobic nature.

Experimental Section Materials

Anhydrous DMF, DMSO,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),1,1′-Carbonyldiimidazole (CDI), N-hydroxysuccinimide (NHS), AZT(azidothymidine), diethylmalonate and other chemicals were purchasedfrom Sigma-Aldrich and used without further purification. Near Infra Reddyes (DiI-D282, DiD-D7757, and DiR-012731) and4′,6-diamidino-2-phenylindole (DAPI-01306) were purchased fromInvitrogen, whereas the EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) wasobtained from Pierce Biotechnology. The folate-receptor-overexpressinghuman lung carcinoma cell line A549 (CCL-185) was obtained from ATCC.Dialysis membranes were obtained from Spectrum Laboratories.Acetonitrile, tetrahydrofuran and other solvents were purchased fromFisher Scientific and used as received, unless otherwise stated.

Instrumentation

Infrared spectra were recorded on a PerkinElmer Spectrum 100 FT-IRspectrometer. UV/Vis spectra were recorded using CARY 300 Bio UV/Visspectrophotometer. Fluorescence spectra were recorded on a NanoLogHoriba jobin Yvon fluorescence spectrophotometer. NMR spectra wererecorded on a MERCURY 300 MHz spectrometer using the TMS/solvent signalas an internal reference. Gel permeation chromatography (GPC) resultswere obtained using JASCO MD 2010 Plus instrument with PD 2020 lightscattering Precision Detector. Thermo gravimetric analyses (TGA) wereperformed on a SETARAM, Mettler TC11 instrument with sample sizes 10-20mg. All the experiments were done using a heating rate of 10° C./min inair. Atomic Force Microscopic (AFM) images were obtained from Dimension3100 Atomic Force Microscope from Veeco Digital Instruments. Confocalimages were taken on a Zeiss Axioskop 2 mot plus confocal microscope.Flow Cytometry experiments were performed using a BD FACS Caliburmultipurpose flow cytometer system from 80 Biosciences. MTT study hasbeen done using BIO-TEK Synergy HT multi-detection microplate reader.Dynamic light scattering (DLS) studies were done using a PDDLS/CoolBatch40T instrument using Precision Deconvolve 32 software and SEM imageswere taken using Jeol 6400F scanning electron microscope. IVISexperiments were done using IVIS 50 imaging system from Xenogen imagingtechnologies. Analytical Thin Layer Chromatography (TLC) was performedon glass plates coated with silica gel GF 254 and are visualized iniodine vapor. Column chromatography was performed using silica gel(100-200 mesh) and the eluant is mentioned in the procedures below foreach case.

Methods Synthesis of 4-bromobutyl acetate (2)

Tetrahydrofuran (12.2 mL, 148.4 mmol) and potassium bromide (21.1 g,176.5 mmol) were added in a 250 mL round bottom flask containing 150 mLacetonitrile. The reaction mixture was cooled to 0° C., followed bydrop-wise addition of acetyl chloride (11 mL, 155.1 mmol). Subsequently,the mixture was brought to room temperature, where it was continuouslystirred for 36 h. The reaction mixture was poured in water and extractedwith ethyl acetate. The organic layer was washed with water, dried overNa₂SO₄, and concentrated to obtain the pure product as a colorlessliquid.

Yield: 24.3 g (85%). bp: >250° C. ¹H NMR (300 MHz, CDCl₃, δ ppm, J Hz):1.79 (m, 2H), 1.92 (m, 2H), 2.03 (s, 3H), 3.46 (t, 2H, J=7.6), 4.08 (t,2H, J=6.7). ¹³C NMR (75 MHz, CDCl₃, δ ppm): 20.87, 27.36, 29.36, 33.03,63.43, 170.95. IR (CHCl₃): 3038, 2926, 1352, 1243, 1052 cm⁻¹.

Synthesis of 2-(4-Acetoxy-butyl)-malonic acid diethyl ester (3)

Compound 3 was prepared by following a previously reported method.{Santra, 2004 #6} Briefly, diethyl malonate 1 (10 g, 62.5 mmol),4-bromobutyl acetate 2 (15.84 g, 81.3 mmol) were placed in a roundbottom flask containing acetonitrile (120 mL) and stirred for 2 min atroom temperature. Then to this, we added potassium carbonate (34.5 g,250.1 mmol) and refluxed for 36 h. Next, the mixture was filtered andthe filtrate was concentrated to obtain a yellow liquid. This wasextracted with ethyl acetate, and washed with water. The organic layerswere combined and dried over Na₂SO₄, and purified by columnchromatography using 4% ethyl acetate in petroleum ether as the eluent.

Yield: 13.02 g (76%). bp: 250° C. ¹H NMR (300 MHz, CDCl₃, δ ppm, J Hz):1.28 (t, 6H, J=7.6), 1.38 (m, 2H), 1.62 (q, 2H, J=7.2), 1.98 (q, 2H,J=7.7), 2.05 (s, 3H), 3.34 (t, 1H, J=7.7), 4.09 (t, 2H, J=6.6), 4.22 (q,4H, J=7.2). ¹³C NMR (75 MHz, CDCl₃, δ ppm): 14.06, 20.79, 23.74, 28.25,28.25, 51.84, 61.27, 63.89, 169.31, 171.11. IR (CHCl₃): 2982, 1728,1463, 1367, 1233, 1151, 1029, and 860 cm⁻¹.

Synthesis of 2-(4-hydroxy butyl)-malonic acid (4)

2-(4-acetoxy-butyl)-malonic acid diethyl ester 3 (5.0 g, 18.25 mmol, seeSection SI in the Supporting Information for the synthesis of compound3) was taken in a 100 mL round bottom flask containing methanol (50 mL)and stirred at room temperature for 2 min. To this was added NaOH (2.1g, 54.74 mmol) in water (7 mL) and stirred at 90° C. for 8 h. Thereaction mixture was shifted to room temperature and acidified (pH 2-3)with the drop-wise addition of dilute hydrochloric acid at roomtemperature with constant stirring. The mixture was then concentrated byusing rotary evaporator and applying vacuum. To this was addedchloroform (50 mL) and Argon gas was bubbled through the solution at 60°C. to remove excess HCl. The mixture was filtered and the filtrate wasconcentrated. This was then purified by column chromatography using 35%ethyl acetate in petroleum ether as eluent.

Yield: 2.31 g (72%). ¹H NMR (300 MHz, CDCl₃, δ ppm, J Hz): 1.41 (m, 2H),1.59 (m, 2H), 1.91 (q, 2H, J₁=7.3, J₂=7.8), 3.37 (t, 1H, J=7.4), 3.64(t, 2H, J=6.5), 5.54 (bs, 1H). ¹³C NMR (75 MHz, CDCl₃, δ ppm): 23.53,28.52, 31.75, 52.64, 62.11, 170.55. IR (CHCl₃): 3507, 2941, 1710, 1626,1459, 1438, 1391, 1198, 1157, 1050, 947, 772, 741, 664 cm⁻¹.

Synthesis of Hyperbranched Polyester (HBPE) 5

The monomer 4 and the catalyst p-toluene sulphonic acid (100:1 molarratio) were taken in a 10 mL round bottom flask and dried under highvacuum followed by the release of vacuum using dry argon gas. Then theflask was slowly heated to 150° C. under argon atmosphere using an oilbath and it was kept at this temperature for 2 h. The evolution of thebyproduct (water vapor) was clearly visible after the sample was heatedat 150° C. The melt was evacuated at 0.2 mm/Hg for 1 h while maintainingthe same polymerization temperature. The polymer was purified bydissolving in DMF and reprecipitating in methanol. This was thencentrifuged, washed with methanol and dried in a high vacuum pump to getpure polymer.

Yield: 65%. ¹H NMR (300 MHz, DMSO-d₆, δ ppm): 1.25 (m, 2H), 1.52 (m,2H), 1.67 (m, 2H), 3.38 (m, 1H), 3.58 (m, 2H), 5.28 (m, 1H). ¹³C NMR (75MHz, DMSO-d₆, δ ppm): 23.82, 28.23, 51.85, 52.63, 65.37, 170.45. IR:2954, 1727, 1458, 1436, 1343, 1218, 1152, 1054, 943, 858, 743, 694 cm⁻¹.TGA: 10% weight loss at 250° C.

Synthesis of Hyperbranched Polyester Amine (HBPE-EDA) 6: CarbodiimideChemistry

The polymer 5 (0.1 g, 0.0025 mmol) was dissolved in anhydrous DMF (1 mL)using vortex mixture and to this was added 1,1-carbonyldiimidazole CDI(0.041 g, 0.25 mmol) in anhydrous DMF (0.1 mL) drop-wise. The reactionmixture was incubated for 2 h at room temperature. To this was thenadded ethylenediamine (0.015 g, 0.25 mmol) in anhydrous DMF (0.4 mL)drop-wise and incubated at room temperature for 24 h. The resultingreaction mixture was then precipitated in methanol, centrifuged anddried in a vacuum pump to get pure aminated polymer.

Yield: 88%. ¹H NMR (300 MHz, DMSO-d₆, δ ppm): 1.27 (m, 2H), 1.55 (m,2H), 1.74 (m, 2H), 2.26 (m, 4H), 2.88 (m, 4H), 3.34 (m, 1H), 3.63 (m,4H), 4.04 (m, 2H). IR: 3245, 2940, 2864, 1725, 1659, 1534, 1435, 1240,1159, 1062, 1021, 952, 929, 826, 749, 704, 663 cm⁻¹.

Synthesis of Clickable Hyperbranched Polyester (HBPE-PA) 7: CarbodiimideChemistry

Similar procedure has been followed as described for the synthesis ofpolymer 6. Instead of ethylenediamine, propargylamine (0.014 g, 0.25mmol) was used as the starting material.

Yield: 80%. ¹H NMR (500 MHz, DMSO-d₆, δ ppm): 1.28 (m, 2H), 1.54 (m,2H), 1.75 (m, 2H), 2.25 (m, 2H), 3.42 (bs, 1H), 3.96 (m, 4H), 4.03 (m,2H). IR: 3121, 2938, 2864, 2698, 2607, 1725, 1664, 1530, 1458, 1437,1388, 1326, 1254, 1158, 1094, 1062, 929, 827, 748, 662 cm⁻¹.

General Procedures for the Synthesis of Functional PolymericNanoparticles Dye-Encapsulating PNPs (8-10): Solvent Diffusion Method.

Different near IR dye (DiI, DiR or DiD) solutions were prepared bymixing 5 μL of the dye aliquot (10 μg/μL) in 250 μL of DMF. The polymers(5, 6 or 7, 0.025 g) were dissolved in 250 μL of anydrous DMF using avortex mixturer and mixed separately with different dye solution. Theresulting polymer-dye mixture in DMF was added drop-wise to deionizedwater (5 mL) with continuous stirring at room temperature forming dyeencapsulated polymeric nanoparticle. The nanoparticle solution wasdialyzed (using 6-8 K molecular weight cut off dialysis bag) three timesagainst deionized water and phosphate buffered saline (PBS) solution.

Paclitaxel (Taxol®) and DiI Co-Encapsulating Polymeric Nanoparticles10d:

Taxol® (5 μL, 1 mg/mL) and DiI dye (5 μL, 10 μg/μL) were taken in anEppendorf Tube® containing propargylated polymer (7, 0.025 g) in 500 μLDMF and followed the solvent diffusion method as described above.

AZT and DiI Co-Encapsulating Polymeric Nanoparticles 10e:

AZT (azidothymidine) was dissolved in DMF to a final concentration of 1mg/mL. The polymers (5 or 7, 0.025 g) were dissolved in 250 μL of DMFusing a vortexer. Subsequently, AZT (5 μL, 1 mg/mL) and DiI (5 μL, 10μg/mL) were added to the polymer solutions, followed by vortexing. Theresulting polymer-AZT-DiI mixture in DMF was added drop-wise todeionized water (5 mL) with continuous stirring at room temperatureforming DiI and AZT co-encapsulating polymeric nanoparticles. Thenanoparticle solutions were dialyzed (using 6-8 K molecular weight cutoff dialysis bag) three times against deionized water and phosphatebuffered saline (PBS) solution.

Synthesis of Aminopropylazide 12

Chloropropyl amine (7.0 g, 75.26 mmol) and sodium azide (14.23 g, 225.81mmol) were taken in a 100 mL round bottom flask containing 40 mL ofdistilled water and heated at 80° C. for 20 h. The reaction mixture wasconcentrated via a rotavapor using high vacuum, and 2 g of KOH was addedto it and then extracted by using diethyl ether. Subsequently, thereaction mixture was dried over anhydrous sodium sulphate andconcentrated. Then, the mixture was purified through flash columnchromatography using 4% ethyl acetate in petrolium ether as an eluant,in order to obtain the pure aminopropylazide.

Yield: 5.1 g (68%). ¹H NMR (300 MHz, CDCl₃, δ ppm): 1.26 (bs, 2H), 1.81(m, 2H), 2.80 (t, 2H), 3.38 (1, 2H). IR (CHCl₃): 3307, 2941, 2089, 1663,1433, 1370, 1259, 1242, 1075, 1026, 818, 760 cm⁻¹.

Synthesis of Azide-Functionalized Folic Acid 13

1,1′-carbonyldiimidazole CDI (0.022 g, 0.014 mmol) was taken in anEppendorf Tube® containing folic acid (0.05 g, 0.011 mmol) in anhydrousDMF (2 mL) and incubated for 2 h at 35° C. To this we addedaminopropylazide (0.014 g, 0.014 mmol) in anhydrous DMF (100 μL) andincubated it for 24 h at room temperature. The reaction mixture was thencentrifuged and washed to remove excess starting materials. Finally, wedissolved the azide-functionalized folic acid in 1 mL of DMF. Thepresence of a band at 2091 cm⁻¹ in the IR spectrum and a UV absorbanceshoulder at 354 nm confirmed the formation of azide-functionalized folicacid.

Yield: 0.05 g (86%). ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 1.61 (m, 2H),1.65 (m, 2H), 1.90 (m, 2H), 2.19 (t, 2H), 2.78 (t, 2H), 4.18 (q, 1H),4.21 (d, 2H), 6.62 (d, 2H), 7.59 (d, 2H), 8.58 (s, 1H). FT-IR (Neat):3024, 2097, 1685, 1603, 1492, 1375, 1291, 1248, 1180, 1122, 1062, 950,844, 755, 696 cm⁻¹.

Synthesis of Folate-Functionalized PNPs 11a-e Click Chemistry

The propargylated polymeric nanoparticles 10a-e (0.025 g, 6×10⁻³ mmol)in bicarbonate buffer (pH=8.5) were taken to an eppendorf containingcatalytic amount of CuI (0.11 μg, 6×10⁻¹⁰ mmol) in 250 μL of bicarbonatebuffer, vortexed for 30 seconds. To this was added azide-functionalizedfolic acid (13, 0.003 g, 6×10⁻² mmol) in DMSO and the reaction wasincubated at room temperature for 12 h. The final reaction mixture waspurified by dialysis using 6-8 K molecular weight cut off dialysis bag,against deionized water and phosphate buffered saline (PBS) solution.The purified functional PNPs (11a-e) were stored in refrigerator forfurther characterization.

Cell Culture and Cell Viability Studies

Lung carcinoma cells (A549) were grown in Kaighn's modification of Ham'sF12 medium (F12K-Cellgro), supplemented with 5% fetal bovine serum(Heat-inactivated FBS-Cellgro), L-glutamine, streptomycin, amphotericinB, and sodium bicarbonate. The cells were maintained at 37° C., 5% CO₂in a humidified incubator. We used the MTT assay in order to assesspotential cytotoxic effects upon in vitro administration of thedrug/dye-encapsulating functional HBPE nanoparticles. Specifically, lungcarcinoma cells (3000 cells/well) were seeded in 96-well plates, andwere incubated with the nanoparticles for 3 hours at 37° C. Then, eachwell was washed three times with 1×PBS and treated with 20 μl MTT (5μg/μl, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide,Sigma-Aldrich) for 2 hours. The resulting formazan crystals weredissolved in acidified isopropanol (0.1 N HCl) and the absorbance wasrecoded at 570 nm and 750 nm (background), using a Synergy HTmulti-detection microplate reader (Biotek). These experiments wereperformed in triplicates.

Cellular Internalization

Initially, in vitro uptake and internalization of the PNPs was assessedthrough fluorescence laser-scanning confocal microscopy, using a ZeissLSM 510 confocal microscope. Specifically A549 cells (10³) wereincubated for the stated time period with the corresponding PNPpreparation in a humidified incubator (37° C., 5% CO₂). Subsequently,the cells were thoroughly washed three times with 1×PBS and fixed with a10% formalin. Nuclear staining with DAPI was performed as recommended bythe supplier. Then, multiple confocal images were obtained, achieving arepresentative view of the cell-PNP interaction. Confirmation of theconfocal studies was facilitated through FACS and IVIS analyses. ForFACS, 10⁵ lung carcinoma cells were incubated for 6 hours with thecorresponding PNP preparation. Then the cells were detached from theculture dish with 0.05% trypsin, and the resulting pellet wasresuspended in 1 mL culture media. The cell suspension underwent flowcytometric analysis, using a BD FACSCalibur system, in order to quantifythe cellular uptake of the synthesized PNPs. For the IVIS analysis, 10⁵lung carcinoma cells were incubated for 6 hours with the correspondingPNP preparation, and then the supernatant was collected in eppendorftubes. Subsequently, we thoroughly washed the cells with 1×PBS anddetached them, as stated above. The resulting pellets were resuspendedin 1 ml culture media. All Eppendorf Tubes® were examined simultaneouslyon a Xenogen IVIS system, using the following filer sets: DsRed (500-550nm/575-650 nm for DiI), Cy5.5 (615-665 nm/695-770 nm for DiD) and ICG(710-760 nm/810-875 nm for DiR). All experiments were performed intriplicates.

In Vitro Drug/Dye Release:

The in vitro drug/dye release studies were carried out using a dynamicdialysis technique at 37° C. Briefly, 100 μL of PNPs (11d) are incubatedwith a porcine liver esterase (20 μL) inside a dialysis bag (MWCO6000-8000), which is then placed in a PBS solution (pH 7.4). The amountof guest (dye or drug) molecules released from the nanoparticle into thePBS solution was determined at regular time intervals by taking 1 mLaliquots from the PBS solution and measuring the fluorescence intensityat 575 nm for DiI and 372 nm for TaxolSS®. The concentration of theeither dye or drug was calculated using a standard calibration curve.The cumulative fraction of release versus time was calculated using thefollowing equation:Cumulative release(%)=[guest]_(t)/[guest]_(total)×100Where [guest]_(t) is the amount of guest released at time t,[guest]_(total) is the total guest present in the guest encapsulatedPNPs.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

REFERENCES CITED

-   [1] M. Fischer, F. Vögtle, Angew. Chem., Int. Ed. Engl. 1999, 38,    884-905.-   [2] Fréthet, J. M. J.; Henmi, M.; Gitsov, I.; Aoshima, S.; Leduc, M.    R.; Grubbs, R. B. Science 1995, 269, 1080-1083.-   [3] Bharathi, P.; Moore, J. S. J. Am. Chem. Soc. 1997, 119,    3391-3392.-   [4] Magnusson, H.; Malmstro{umlaut over ( )}m, E.; Hult, A.    Macromolecules 2000, 33, 3099-3104.-   [5] Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.;    Sakurai, Y. J. Controlled Release 1993, 24, 119-132.-   [6] Fréchet, J. M. J.; Tomalia, D. A.; Dendrimers and Other    Dendritic Polymers; John Wiley: New York, 2002.-   [7] Flory, P. J. J. Am. Chem. Soc. 1952, 74, 2718.-   [8] Chu, F.; Hawker, C. J. Polym. Bull. 1993, 30, 265.-   [9] Stiriba, S-E.; Kautz, H.; Frey, H. J. Am. Chem. Soc. 2002, 124,    9698.-   [10] Plummer, C. J. G.; Garamszegi, L.; Leterrier, Y.; Rodlert, M    and Manson, J-A. E. Chem. Mater. 2002, 14, 486-488.-   [11] Schmid, G. Chem. Rev. 1992, 92, 1709.-   [12] Fonseca, C.; Simoes, S.; Gaspar, R. J. Controlled Release 2002,    83, 273-286.-   [13] Gupte, A.; Ciftci, K. Int. J. Pharm. 2004, 276, 93-106.-   [14] Sparreboom, A.; Baker, S. D.; Verweij, J. J. Clin. Oncol. 2005,    23, 7765-7767.-   [15] J. R. McCarthy, J. M. Perez, C. Bruckner, R. Weissleder, Nano    Lett. 2005, 5, 2552-2556.-   [16] B. S. Packard, D. E. Wolf, Biochemistry 1985, 24, 5176-5181.-   [17] Mitra, A.; Lin, S. J. Pharm. Pharmacol. 2003, 55, 895-902.-   [18] E. Y. Sun, L. Josephson, R. Weissleder, Molecular Imaging 2006,    5, 122-128.-   [19] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed.    Engl. 2001, 40, 2004-2021;-   [20] M. A. White, J. A. Johnson, J. T. Koberstein, N. J. Turro, J.    Am. Chem. Soc. 2006, 128,    -   11356-11357.

That which is claimed is:
 1. A polymer comprising the repeating unit:


2. A polymeric nanoparticle comprising the polymer of claim
 1. 3. Thepolymeric nanoparticle of claim 2, wherein the polymeric nanoparticle isbiodegradable.
 4. The polymeric nanoparticle of claim 2, having one ormore internal hydrophobic pockets and a hydrophilic outer surface. 5.The polymeric nanoparticle of claim 2, having an average diameter of115±25 nm.
 6. The polymeric nanoparticle of claim 2, being spherical inshape.
 7. The polymeric nanoparticle of claim 2, having a positive zetapotential.
 8. The polymeric nanoparticle of claim 2, further comprisinga hydrophobic near-infrared fluorescent dye encapsulated therein.
 9. Thepolymeric nanoparticle of claim 8, wherein the dye is selected from thegroup consisting of DiI, DiR, DiD, and combinations thereof.
 10. Thepolymeric nanoparticle of claim 2, further comprising a therapeutic drugencapsulated therein.
 11. The polymeric nanoparticle of claim 10,wherein the therapeutic drug comprises an anti-cancer drug.
 12. Thepolymeric nanoparticle of claim 10, wherein the therapeutic drugcomprises azidothymidine.
 13. The polymeric nanoparticle of claim 10,wherein the therapeutic drug comprises paclitaxel.
 14. The polymericnanoparticle of claim 10, further comprising a fluorescent dyeco-encapsulated with said therapeutic drug.
 15. An aqueous suspension ofthe polymeric nanoparticle of claim
 2. 16. The aqueous suspension ofclaim 15, further comprising a hydrophobic near-infrared fluorescentdye.
 17. The aqueous suspension of claim 15, further comprising atherapeutic drug.
 18. The aqueous suspension of claim 15, furthercomprising a hydrophobic near-infrared fluorescent dye and a therapeuticdrug.